The preparation of bonded phases to be used as the stationary phase for chromatographic applications has been widely studied. Silanes are the most commonly used surface modifying reagents to prepare bonded phases in liquid chromatography. The chemistry of silanes with various surfaces is well studied. A general discussion of the reaction of silanes with the surface of silicaceous chromatographic support materials is provided in HPLC Columns: Theory, Technology, and Practice, U. D. Neue, Wiley-VCH, Inc., New York (1997). Additional details on the reaction of silanes with porous silicas are disclosed in Characterization and Chemical Modification of the Silica Surface, E. F. Vansant, et al., Elsevier Science B. V. New York (1995). A broad description of the reactions of silanes with a variety of materials is given in Silica Gel and Bonded Phases, Their Production, Properties and Use in LC, R. P. W. Scott, John Wiley & Sons, New York (1993).
There are numerous improvements in the art of bonded phases. These improvements are aimed at producing specialized phases useful in normal phase chromatography, for separating polar analytes, or reverse phase chromatography for separating nonpolar analytes, solving problems relating to stability in high or low pH mobile phase conditions, providing phases that are useful under normal phase as well as reversed phase mobile phase conditions. Other problems stem from difficulties in creating bonded phases that provide for reduced residual silanol residues, and further, that do not exhibit hydrolysis of the bonded phase. Some investigators have approached the latter problem by including bulky substituents on the silane to provide steric protection against hydrolysis for the bonded phase. For example, U.S. Pat. No. 4,705,725 to Glajch describes bonding a monofunctional silane to a substrate wherein the silane contains at least two sterically-protecting groups attached to the silicon atom of the silane.
Specialized phases have been produced for both reversed phase and normal phase chromatography. For normal phase chromatography, silica has been coated with salts of heavy metals to produce a silica having unique selectivities for analytes that form complexes with the metal ions. For example, silver has been coated onto silica to be utilized in argentation chromatography. (See, U. D. Neue, HPLC Columns, Theory, Technology, and Practice, Wiley-VCH, New York, 1997, Chapter 9, page 164-182). In addition, silica supports impregnated with transition metals are known in the catalyst art for treatment of the products of combustion (See, e.g., U.S. Pat. No. 5,990,039 to Paul, et al.).
U.S. Pat. No. 5,393,892 to Krakowiak, et al. describes the selective removal of alkali, alkaline earth metal, Pb and Tl cations from complex matrices containing these ions by means of a crown ether covalently bonded through a spacer to a support material consisting of sand, silica gel, glass, glass fibers, alumina, nickel oxide, zirconia or titania. However, this patent does not concern phases for chromatographies. The material was used for the removal of alkali, alkaline earth metal, Pb and Tl cations, and not for analytical or chromatographic purposes.
Useful bonded phases for use in both normal and reverse phase chromatography include the use of the polar cyano functionality, and cyanopropyl bonded phase columns have been widely applied in the separation of analytes. For example, Okusa, K., et al. (2000) J Chromatogr. 869, 143-149 described a cyanopropyl bonded column used to separate polar compounds from biological samples. The column reportedly showed specific selectivity and suitability for use in both normal and reversed phase high performance liquid chromatography (HPLC). Okusa, et al. reported that both the separating selectivity and durability of the cyanopropyl bonded phase are dependent on the preparation conditions, and that the irreversible adsorption of compounds is related to the density of cyanopropyl groups on the silica support. Non-endcapped bonded phases were reported to inhibit adsorption of basic compounds when a high loading of cyanopropyl groups was utilized.
Although cyano columns are commercially available and have a number of advantages, their use is not very widespread due to the problems of poor chemical and mechanical stability (see Okusa, et al. and U. D. Neue, HPLC Columns, Theory, Technology, and Practice, Wiley-VCH, New York, 1997, Chapter 9, page 164-182). For example, cyano columns typically are ascribed poor retention time stability in low pH mobile phases due to acid induced ligand hydrolysis from the silica support. In addition, stability in high pH mobile phases can suffer due to dissolution of the silica skeleton, where the short chain ligand provides little protection. In both these instances, there is a change in the chromatographic profile (J. E. O'Gara, J. E., et al. (2000) J Chromatogr. 893, 245-251). Frequently the pH must be maintained at a prescribed level, or the column undergoes irreversible damage and loses its efficiency and characteristics, such as the ability to produce narrow peaks, desirable retention volumes or resolve components of a mixture. This damage can occur even if the accidental use outside the narrow pH range defined for the column is only for a short period of time.
Another deficiency reported for cyanopropyl columns is their tendency to exhibit phase collapse in solvents of intermediate polarity. The instability is mechanical in nature, not chemical, in which the packed bed collapses into a more dense state. The collapse phenomenon is akin to the events that occur when a bucket full of wet beach sand is tapped from the side, the sand settles, and water rises to the surface (see U. D. Neue, HPLC Columns, Theory, Technology, and Practice, Wiley-VCH, New York, 1997, Chapter 9, page 164-182.). In both nonpolar and polar solvents, this catastrophic collapse is prevented by the adhesion of particles to each other. In solvents of intermediate polarity, this adhesion vanishes and bed collapse can occur.
Another disadvantage of cyano bonded phase relates to polar interactions with analytes. It is unclear how much of the polar interaction of this packing is due to the cyano functional group and how much is due to residual silanols. Most likely, both participate in the retention of analytes. Detailed studies of the retention mechanism of this bonded phase are not available.
One attempt to solve the problem of polar interactions has been to introduce a bulkier substituent on the silicon atom of the silane reagent in place of the methyl groups, as described in U.S. Pat. No. 4,705,725 to Glajch, et al. This patent discloses that a (3-cyanopropyl)diisopropylsiloxane bonded phase has improved hydrolytic stability, which is apparently due to steric protection of the siloxane bond by the large isopropyl side groups.
However, there remains a need in the art to provide a stable and reproducible cyano bonded phase suitable for chromatographic separations. There is a further need in the art to provide a cyano bonded phase that is not susceptible to phase collapse. There is an additional need in the art to provide a cyano bonded phase that resists hydrolysis in base or acid conditions. There is a further need in the art to provide a cyano bonded phase that can provide superior and rapid separations between basic compounds.